PROJECT SUMMARY Regulatory T (Treg) cells, characterized by the expression of FOXP3, are a distinct lineage of T lymphocytes that control immunological self-tolerance and tissue homeostasis. These cells are associated with a multitude of human diseases, such as autoimmune disease, inflammation, organ transplant rejection, and some infections and cancers. The biomedical community has developed an intense interest in targeting Treg cells for immunotherapy; however, significant barriers, for example Treg-cell functional heterogeneity and instability, need to be surmounted in order to achieve clinical feasibility. The proposed study aims to understand a novel molecular mechanism that control the stability and function of Treg cells. Our preliminary data have found that a novel O-linked N-Acetylglucosamine (O-GlcNAc) protein modification, activated by T-cell receptor (TCR) signaling, is indispensable for the suppressive function of Treg cells. Treg cell-specific knockout of O-GlcNAc transferase (OGT), the enzyme catalyzing O-GlcNAcylation, does not affect Treg cell lineage specification but results in the development of an aggressive autoimmune syndrome in mice. O-GlcNAcylation modifies and stabilizes FOXP3 protein and transcriptomic analyses have revealed that IL-2/STAT5 signaling is suppressed in OGT-deficient Treg cells. Therefore, we hypothesize that TCR-initiated protein O-GlcNAcylation modulates FOXP3 and STAT5, thereby linking TCR and IL-2 signaling pathways, to regulate Treg cell homeostasis and effector function. In Aim 1, we hypothesize that loss of O-GlcNAcylation destabilizes FOXP3 protein, eliminating the suppressive function of Treg cells and converting Treg cells into effector T cells. We will determine whether O-GlcNAcylation counteracts ubiquitination to stabilize FOXP3 protein, thus maintaining the Treg identity. By generating FOXP3 mutant mice, in which O-GlcNAcylation is specifically abolished, we will test whether FOXP3 O-GlcNAcylation is required for the suppressive activity of Treg cells. In Aim 2, we propose to examine the effect of STAT5 O-GlcNAcylation on Treg cell suppressive function. We will test the hypothesis that O-GlcNAcylation controls IL-2-stimulated STAT5 activation in Treg cells and FOXP3 and STAT5 act cooperatively to mediate the effect of protein O-GlcNAcylation in Treg cell function. In Aim 3, we will identify the molecular link between TCR and O-GlcNAcylation in Treg cells. We will test the hypothesis that TCR signaling promotes protein O- GlcNAcylation and Treg cell suppressive activity in a Ca2+/CaMKII-dependent manner. Lastly, through unbiased O-GlcNAc proteomics, we will characterize TCR-regulated protein O-GlcNAcylation profile and identify potential complementary mechanisms for O-GlcNAc-mediated Treg cell function. The proposed study will stimulate the opening of a new avenue in therapeutics directed to activate and maintain Treg cells for immunotherapy.